RESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated cardiomyopathy (DCM). We have previously shown that DMD (mdx) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca2+) cycling related to early-stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age-dependent DCM. METHODS: Here, we used hiPSC-CMs from DMD patients selected by Speckle-tracking echocardiography and canine DMD cardiac biopsies to assess key early-stage Duchenne DCM features. RESULTS: Dystrophin deficiency was associated with RyR2 remodelling and SR Ca2+ leak (RyR2 Po of 0.03 ± 0.01 for HC vs. 0.16 ± 0.01 for DMD, P < 0.01), which led to early-stage defects including senescence. We observed higher levels of senescence markers including p15 (2.03 ± 0.75 for HC vs. 13.67 ± 5.49 for DMD, P < 0.05) and p16 (1.86 ± 0.83 for HC vs. 10.71 ± 3.00 for DMD, P < 0.01) in DMD hiPSC-CMs and in the canine DMD model. The fibrosis was increased in DMD hiPSC-CMs. We observed cardiac hypocontractility in DMD hiPSC-CMs. Stabilizing RyR2 pharmacologically by S107 prevented most of these pathological features, including the rescue of the contraction amplitude (1.65 ± 0.06 µm for DMD vs. 2.26 ± 0.08 µm for DMD + S107, P < 0.01). These data were confirmed by proteomic analyses, in particular ECM remodelling and fibrosis. CONCLUSIONS: We identified key cellular damages that are established earlier than cardiac clinical pathology in DMD patients, with major perturbation of the cardiac ECC. Our results demonstrated that cardiac fibrosis and premature senescence are induced by RyR2 mediated SR Ca2+ leak in DMD cardiomyocytes. We revealed that RyR2 is an early biomarker of DMD-associated cardiac damages in DMD patients. The progressive and later DCM onset could be linked with the RyR2-mediated increased fibrosis and premature senescence, eventually causing cell death and further cardiac fibrosis in a vicious cycle leading to further hypocontractility as a major feature of DCM. The present study provides a novel understanding of the pathophysiological mechanisms of the DMD-induced DCM. By targeting RyR2 channels, it provides a potential pharmacological treatment.
Assuntos
Cardiomiopatias , Cardiomiopatia Dilatada , Humanos , Camundongos , Animais , Cães , Cardiomiopatia Dilatada/etiologia , Distrofina/genética , Distrofina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Camundongos Endogâmicos mdx , Cálcio/metabolismo , Proteômica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , FibroseRESUMO
BACKGROUND: Duchenne muscular dystrophy is associated with progressive deterioration in left ventricular (LV) function. The golden retriever muscular dystrophy (GRMD) dog model recapitulates the pathology and clinical manifestations of Duchenne muscular dystrophy. Importantly, they develop progressive LV dysfunction starting at early age. OBJECTIVES: The authors tested the cardioprotective effect of chronic administration of the ARM036, a small molecule that stabilizes the closed conformation of the cardiac sarcoplasmic reticulum ryanodine receptor/calcium release channel (RyR2) in young GRMD-dogs. METHODS: Two-month-old GRMD-dogs were treated with ARM036 or placebo for 4 months. Healthy-dogs of the same genetic background served as controls. Cardiac function was evaluated by conventional and 2-dimensional speckle-tracking echocardiography. Cardiac cellular and molecular analyses were performed at 6 months old. RESULTS: Conventional echocardiography showed normal LV dimensions and ejection fraction in 6-month-old GRMD dogs. Interestingly, 2-dimensional speckle-tracking echocardiography revealed decreased global longitudinal strain and the presence of hypokinetic segments in placebo-treated GRMD dogs. Single-channel measurements revealed higher RyR2 open probability at low resting Ca2+ in GRMD cardiomyocytes than in controls. ARM036 prevented those in vivo and in vitro dysfunctions in GRMD dogs. Myofilament Ca2+-sensitivity was increased in permeabilized GRMD cardiomyocytes at short sarcomere length. ARM036 had no effect on this parameter. Cross-bridge cycling kinetics were altered in GRMD myocytes and recovered with ARM036 treatment, which coincided with the level of myosin binding protein-C-S glutathionylation. CONCLUSIONS: GRMD-dogs exhibit early LV dysfunction associated with altered myofilament contractile properties. These abnormalities were prevented pharmacologically by stabilizing RyR2 with ARM036.
Assuntos
Distrofia Muscular de Duchenne/complicações , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Disfunção Ventricular Esquerda/etiologia , Função Ventricular Esquerda/fisiologia , Animais , Biópsia , Modelos Animais de Doenças , Cães , Ecocardiografia , Distrofia Muscular de Duchenne/diagnóstico , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miofibrilas/metabolismo , Miofibrilas/patologia , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patologia , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologiaRESUMO
Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.
Assuntos
Cardiomiopatia Dilatada/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Envelhecimento/genética , Envelhecimento/patologia , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Dano ao DNA/genética , Modelos Animais de Doenças , Distrofina/deficiência , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Células-Tronco/metabolismo , Células-Tronco/patologiaAssuntos
Pesquisa Biomédica/história , Cardiologia/história , Insuficiência Cardíaca/história , Diester Fosfórico Hidrolases/história , Escolha da Profissão , AMP Cíclico/história , AMP Cíclico/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/fisiopatologia , História do Século XX , História do Século XXI , Humanos , Isoenzimas , Diester Fosfórico Hidrolases/metabolismo , Sistemas do Segundo MensageiroRESUMO
BACKGROUND: While mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to exercise-induced or catecholaminergic polymorphic ventricular tachycardia (CPVT), its association with polymorphic ventricular tachycardia (PMVT) occurring at rest is unclear. We aimed at constructing a patient-specific human-induced pluripotent stem cell (hiPSC) model of PMVT occurring at rest linked to a single point mutation in RyR2. METHODS: Blood samples were obtained from a patient with PMVT at rest due to a heterozygous RyR2-H29D mutation. Patient-specific hiPSCs were generated from the blood samples, and the hiPSC-derived cardiomyocytes (CMs) were generated via directed differentiation. Using CRIPSR/Cas9 technology, isogenic controls were generated by correcting the RyR2-H29D mutation. Using patch-clamp, fluorescent confocal microscopy and video-image-based analysis, the molecular and functional properties of RyR2-H29D hiPSCCMs and control hiPSCCMs were compared. FINDINGS: RyR2-H29D hiPSCCMs exhibit intracellular sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing. RyR2-H29D enhances the contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) that exacerbates abnormal Ca2+ release in RyR2-H29D hiPSCCMs. RyR2-H29D hiPSCCMs exhibit shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. The RyR2-H29D mutation causes post-translational remodeling that is fully reversed with isogenic controls. INTERPRETATION: To conclude, in a model based on a RyR2 point mutation that is associated with short-coupled PMVT at rest, RyR2-H29D hiPSCCMs exhibited aberrant intracellular Ca2+ homeostasis, shortened action potentials, arrhythmias and abnormal contractile properties. FUNDING: French Muscular Dystrophy Association (AFM; project 16,073, MNM2 2012 and 20,225), "Fondation de la Recherche Médicale" (FRM; SPF20130526710), "Institut National pour la Santé et la Recherche Médicale" (INSERM), National Institutes of Health (ARM; R01 HL145473) and New York State Department of Health (NYSTEM C029156).
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/terapia , Alelos , Sistemas CRISPR-Cas , Cálcio/metabolismo , Sinalização do Cálcio , Genótipo , Homeostase , Humanos , Imuno-Histoquímica , Mutação , Processamento de Proteína Pós-Traducional , Transplante de Células-Tronco , Taquicardia Ventricular/etiologiaRESUMO
The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.
Assuntos
Doença de Alzheimer/metabolismo , Cálcio/metabolismo , Transtornos Cognitivos/metabolismo , Processamento de Proteína Pós-Traducional , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Doença de Alzheimer/patologia , Animais , Sinalização do Cálcio , Transtornos Cognitivos/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Fosforilação , Reconhecimento Psicológico/fisiologia , Retículo Sarcoplasmático/metabolismoRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. In the last decade, number of evidences showing miRNAs contribution to the regulation of apoptosis, cellular proliferation, differentiation, and other important cellular processes is constantly growing. Specific miRNA expression signatures have been identified in variety of human cancers as well as pathologies of cardiovascular and urinary systems. Our chapter focuses on the potential of urinary miRNAs to serve as biomarkers in uro-oncology, nephrology, and cardiology. We discuss in detail recent knowledge about the origin of urinary miRNAs, their stability, quality control, and their utility as a potential new class of biomarkers in medicine. Finally, we summarize the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in urologic cancers, nephrology, and cardiology.
Assuntos
Cardiopatias/urina , MicroRNAs/urina , Neoplasias/urina , Doenças Urológicas/urina , Biomarcadores/urina , Feminino , Cardiopatias/diagnóstico , Cardiopatias/genética , Cardiopatias/patologia , Humanos , Masculino , MicroRNAs/genética , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologia , Estabilidade de RNA , Doenças Urológicas/diagnóstico , Doenças Urológicas/genética , Doenças Urológicas/patologiaRESUMO
AIMS: TRPM2 is a Ca(2+)-permeable cationic channel of the transient receptor potential (TRP) superfamily that is linked to apoptotic signalling. Its involvement in cardiac pathophysiology is unknown. The aim of this study was to determine whether the pro-apoptotic cytokine tumour necrosis factor-α (TNF-α) induces a TRPM2-like current in murine ventricular cardiomyocytes. METHODS AND RESULTS: Adult isolated cardiomyocytes from C57BL/6 mice were exposed to TNF-α (10 ng/mL). Western blotting showed TRPM2 expression, which was not changed after TNF-α incubation. Using patch clamp in whole-cell configuration, a non-specific cation current was recorded after exposure to TNF-α (ITNF), which reached maximal steady-state amplitude after 3 h incubation. ITNF was inhibited by the caspase-8 inhibitor z-IETD-fmk, the antioxidant N-acetylcysteine, and the TRPM2 inhibitors clotrimazole, N-(P-amylcinnamoyl) anthranilic acid and flufenamic acid (FFA). TRPM2 has previously been shown to be activated by ADP-ribose, which is produced by poly(ADP-ribose) polymerase 1 (PARP-1). TNF-α exposure resulted in increased poly-ADP-ribosylation of proteins and the PARP-1 inhibitor 3-aminobenzamide inhibited ITNF. TNF-α exposure increased the mitochondrial production of reactive oxygen species (ROS; measured with the fluorescent indicator MitoSOX Red), and this increase was blocked by the caspase-8 inhibitor z-IETD-fmk. Clotrimazole and TRPM2 inhibitory antibody decreased TNF-α-induced cardiomyocyte death. CONCLUSION: These results demonstrate that TNF-α induces a TRPM2 current in adult ventricular cardiomyocytes. TNF-α induces caspase-8 activation leading to ROS production, PARP-1 activation, and ADP-ribose production. TNF-induced TRPM2 activation may contribute to cardiomyocyte cell death.
Assuntos
Caspase 8/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPM/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Clotrimazol/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Técnicas de Patch-Clamp , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPM/antagonistas & inibidoresRESUMO
BACKGROUND: Disruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies. METHODS: To test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in ß-sarcoglycan (Sgcb-/-). RESULTS: Skeletal muscle RyR1 from Sgcb-/- deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb-/- deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb-/- mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy. CONCLUSIONS: Our data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies.
RESUMO
Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (3-6 months) adults. This RyR1 channel complex remodeling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca(2+) release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function.
Assuntos
Envelhecimento , Cálcio/metabolismo , Debilidade Muscular/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcopenia/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/sangue , Proteínas de Ligação a Tacrolimo/deficiência , Proteínas de Ligação a Tacrolimo/metabolismo , Tiazepinas/farmacologiaRESUMO
Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.
Assuntos
Caspase 8/metabolismo , Ventrículos do Coração/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Ativação Enzimática , Fluorescência , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Fenantridinas/metabolismo , Ratos , Ratos Endogâmicos WKY , Fator de Necrose Tumoral alfa/sangue , Remodelação VentricularRESUMO
RATIONALE: Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young individuals during ß-adrenergic stimulation. OBJECTIVE: This study sought to determine the effects of a novel RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca(2+)). METHODS AND RESULTS: Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2. RyR2 channels were measured to examine the regulation of the channels by cytosolic versus luminal sarcoplasmic reticulum Ca(2+). A 50-year-old white man with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. cAMP-dependent protein kinase-phosphorylated RyR2-G230C channels exhibited a significantly higher open probability at diastolic Ca(2+) concentrations, associated with a depletion of calstabin2. The luminal Ca(2+) sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like. CONCLUSIONS: The RyR2-G230C mutant exhibits similar biophysical defects compared with previously characterized CPVT mutations: decreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca(2+) dependence for activation under conditions that simulate exercise, consistent with a "leaky" channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca(2+) activation. Thus, diastolic sarcoplasmic reticulum Ca(2+) leak caused by reduced calstabin2 binding and a leftward shift in the Ca(2+) dependence for activation by diastolic levels of cytosolic Ca(2+) is a common mechanism underlying CPVT.
Assuntos
Cálcio/fisiologia , Morte Súbita Cardíaca , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Taquicardia Ventricular/fisiopatologia , Catecolaminas/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Citosol/fisiologia , Diástole/fisiologia , Eletrocardiografia , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Fenótipo , Mutação Puntual , Proteínas Recombinantes/genética , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/genética , Proteínas de Ligação a Tacrolimo/fisiologiaRESUMO
Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, ß-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of ß-blocker therapy in HF.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Insuficiência Cardíaca/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Substituição de Aminoácidos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/genética , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutação de Sentido Incorreto , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismoRESUMO
Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane beta-barrel domain that is occluded by an N-terminal alpha-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a beta-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
In Gram-negative bacteria and eukaryotic organelles, beta-barrel proteins of the outer membrane protein 85-two-partner secretion B (Omp85-TpsB) superfamily are essential components of protein transport machineries. The TpsB transporter FhaC mediates the secretion of Bordetella pertussis filamentous hemagglutinin (FHA). We report the 3.15 A crystal structure of FhaC. The transporter comprises a 16-stranded beta barrel that is occluded by an N-terminal alpha helix and an extracellular loop and a periplasmic module composed of two aligned polypeptide-transport-associated (POTRA) domains. Functional data reveal that FHA binds to the POTRA 1 domain via its N-terminal domain and likely translocates the adhesin-repeated motifs in an extended hairpin conformation, with folding occurring at the cell surface. General features of the mechanism obtained here are likely to apply throughout the superfamily.